Optical projection tomography

ABSTRACT

Apparatus for obtaining an image of a specimen ( 6 ) by optical projection tomography comprises a light scanner, such as a light-scanning confocal microscope ( 1, 2, 3 ) for subjecting the specimen ( 6 ) to a scanning movement of incident light.

FIELD OF THE INVENTION

This invention relates to optical projection tomography.

BACKGROUND TO THE INVENTION

Optical projection tomography is a technique for producingthree-dimensional images of specimens, one example being disclosed inthe applicant's specification WO 02/095476. The invention aims toprovide a different way of directing the light onto the specimen,particularly in the case of fluorescent imaging, with a view to reducingnoise or interference in the series of images and providing improveddepth of focus in the series of images.

SUMMARY OF THE INVENTION

According to one aspect of the invention there is provided apparatus forobtaining an image of a specimen by optical projection tomography, theapparatus comprising light-scanning means and a rotary stage forrotating the specimen to indexed positions in each of which the specimenis in use subjected to a scanning movement of incident light by thescanning means.

The incident light may be scanned in a direction perpendicular to anoptical axis defined by the light passing through the apparatus.

The light scanning means may form part of a confocal scanningmicroscope.

According to another aspect of the invention there is provided a methodof obtaining an image of a specimen by optical projection tomograpy, themethod comprising scanning the specimen with a light beam and detectinglight emanating from the specimen to derive the image.

Preferably, the detector detects light which exits or by-passes thespecimen parallel to the beam incident on the specimen.

The incident light is preferably scanned in a raster pattern, onecomplete scan being undertaken at each indexed position of the specimen.

According to the present invention, samples for use in the presentinvention may be prepared as described in the earlier patentapplications and/or employing conventional pathological and histologicaltechniques and procedures well known to persons skilled in the art.

For example, in-situ hybridisation (particularly useful for detectingRNAs):Hammond K L, Hanson I M, Brown A G, Lettice L A, Hill R E“Mammalian and Drosophila dachsund genes are related to the Skiproto-oncongene and are expressed in eye and limb”. Mech Dev. 1998June;74(1-2):121-31.

Immunohistochemistry (particularly useful for detecting proteins andother molecules): Sharpe J, Ahlgren U, Perry P, Hill B, Ross A,Hecksher-Sorensen J, Baldock R, Davidson D. “Optical projectiontomography as a tool for 3D microscopy and gene expression studies”Science. 2002 Apr. 19;296(5567):541-5.

It will be appreciated that modification may be made to the inventionwithout departing from the scope of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention will now be described, by way of example, with referenceto the accompanying drawings, in which:

FIG. 1 is a diagram of the apparatus forming the preferred embodiment ofthe invention,

FIGS. 2 a and 2 b show how the microscope optics of the apparatus can bearranged to have low numerical aperture or high numerical aperture,

FIG. 3 shows known image-forming optics,

FIGS. 4 and 5 show the image-forming optics of an optical system of theinventive apparatus,

FIGS. 6 a, 6 b, 6 c and 6 d show representative light paths for theoptical system of the inventive apparatus,

FIGS. 7 a, 7 b and 7 c illustrate how different degrees of refractionaffect operation of the optical system,

FIG. 8 illustrates how refraction is measured using a one-dimensionalarray of detectors, and

FIGS. 9 to 12 illustrate, in three dimensions, the operation of theoptical system.

DETAILED DESCRIPTION OF THE DRAWINGS

Referring to FIG. 1, the apparatus comprises a light source 1 (in theform of a laser) which supplies light to a two-dimensional lightscanning means 2, the scanning mechanism of which has a dual mirrorsystem. Light with a scanning motion is fed through image-forming optics3. A dichroic mirror 4 interposed between the light source 1 and thescanning means 2 directs returned light to a high speed light detector5. The components 1 to 5 may be provided by a confocal light-scanningmicroscope.

Light from the optics 3 passes through a specimen 6 which is rotatedwithin, and supported by, a rotary stage 7 which in structurecorresponds to the rotary stage disclosed in the applicant's co-pendingInternational Patent Application No. PCT/GB02/02373. The rotary stage 7rotates the specimen 6 to successive indexed positions at each of whichone complete scan of the excitation light is undertaken whilst thespecimen is stationary. After passing through the specimen 6, the lightis processed by an optical system 8 which directs the light to aone-dimensional or two-dimensional array of high speed light detectors9.

In fluorescence mode, light from the specimen 6 is returned through theoptics 3 and the scanning means 2 and thence, via the mirror 4, to thehigh speed light detector 5. In this method of fluorescence imaging, theexcitation light enters one side of the specimen and leaves the specimenfrom the same side thereof before being detected. It is in thetransmission mode, to be described, that the components shown to theright of the stage 7 in FIG. 1 are used.

The microscope optics 3 may have a high numerical aperture (FIG. 2 a) ormay be adapted to have a low numerical aperture (FIG. 2 b) which isuseful for some specimens to be imaged.

FIG. 3 illustrates a known image-forming system. The light from anypoint on the focal plane 12 (within the specimen) is collected andrefracted by a lens 13 towards a single point in the image plane 14.There exists a symmetry such that any point on the image plane 14 mapsto a point in the focal plane 12 and vice versa.

By contrast, the need for an image-forming optical arrangement isremoved in the inventive “non-focal” optics of FIGS. 4 and 5 whichdisplays no such symmetry. The non-focal optical system 8 is representedby a convex lens 15. The light from a single point on the focal plane 12is not focussed onto a single light detector. It is diverged such thatonly the light which exits or by-passes the specimen 6 parallel to theincident beam reaches the single light detector 9 a positioned on theoptical axis. The purpose of the lens 15 in FIGS. 4 and 5 is differentfrom FIG. 3. It functions in a light-scanning situation. The light beamis scanned (e.g. in a raster pattern) across the specimen through amultitude of different positions (five of which are illustrated as theblack arrows in FIG. 5). The purpose of the non-focal optical system 8(i.e. the lens 15) is to direct onto the single light detector 9 a,light which exits or by-passes the specimen parallel to the incidentbeam, irrespective of the scanning position of the light beam. Inspecimens which cause significant scattering of light the system allowsa higher signal-to-noise ratio to be obtained by limiting detection ofscattering light.

FIGS. 6 a to 6 d, which illustrate scattering as an example to showdeviation from the original beam position, illustrate somerepresentative light paths for rays (derived from a laser beam) emittedfrom the specimen 6 while passing through the non-focal optical system.The beam approaching the specimen from the left is the beam incident onthe specimen.

In FIG. 6 a rays scattered from a point in the centre of the specimen 6are diverged away from the light detector 9 a. The proportion ofscattered rays which are detected can be adjusted by changing theeffective size of the detector. An adjustable iris allows this control(which is very similar to the pin-hole in a scanning confocalmicroscope). Alternatively, the position of the lens can be adjusted tocause more or less divergence of the scattered rays. In opticalimage-forming systems, an airy disc is the interference pattern producedby the light emitted from a single point within the specimen. Opticalsystems which produce larger airy discs have lower resolving power, asairy discs from neighbouring points within the specimen will overlap.The concept of the airy disc is not strictly relevant to aprojection-measuring system like this, however a similar concept doesexist. In the case of the non-focal optics described here, light fromeach projection creates a very broad distribution of intensities (at theposition of the detector) similar to a broad airy disc, which mightsuggest low resolving power. However, as only a single projection ismeasured at any one time even very broad distributions cannot interferewith each other.

In FIG. 6 b rays scattered from other points along the same line sampledin FIG. 6 a, are also diverged away from the light detector 9 a.

In FIG. 6 c unscattered light from a different scanned position (blackarrow) is emitted from the specimen 6 substantially parallel to theoptical axis, and is therefore refracted towards the light detector 9 a.As in FIGS. 6 a and 6 b, scattered light is directed away from thedetector 9 a.

In FIG. 6 d unscattered rays from any scanned position are directed ontothe light detector 6. The arrows represent successive positions of thelaser beam as it is scanned across the specimen 6 in a directionperpendicular to the optical axis.

All experiments done so far with optical projection tomography have hadto assume that although some of the light is scattered, the refractiveindex of the specimen is uniform. Recent experiments have demonstratedthat a number of important specimens (including medical imaging ofbiopsies) display non-uniform refractive indexes. This means that thecurrent algorithms are not accurately imaging the specimen—distortionsand artefacts are introduced. The apparatus described reduces thisproblem by measuring information not previously available relating tothe angle at which a light beam exits from the specimen. In general, inspecimens with low scattering but non-uniform distribution of refractiveindex the system allows this non-uniform distribution to be calculatedby measuring the degree of refraction experienced by each projection.

In the use of the present apparatus a clearing agent (such as BABB) isused such that the majority of the light is not scattered. It is howeversubject to a different form of disruption—refraction. In FIG. 7,scattered light is indicated by broken lines, while the main path oflight is shown as a solid line. In the first example of FIG. 7 a thispath is not bent as it passes through the specimen 6 (it is onlyrefracted on passing through the lens). The main path does pass througha region of the specimen with a higher refractive index than the rest(grey disc), however both the interfaces it encounters between regionsof differing refractive index are perpendicular to the light path, so norefraction occurs.

In the second case of FIG. 7 b, the illumination beam is slightly higherand therefore the interfaces it encounters between the grey region andthe white region of the specimen (different refractive indexes) areslightly displaced from perpendicular. This causes two slightrefractions of the main path such that when the light emerges from thespecimen it is no longer parallel to the incident beam and is directedslightly to the side of the original central light detector 9 a. Ifauxiliary light detectors 9 b are positioned on either side of thecentral detector 9 a, these can measure the degree of refraction. Anyprojection will give a certain distribution of intensities along thearray of light detectors. The distribution of intensities can be used todetermine the angle at which the main light path emerged from thespecimen. The system need only determine where the centre of thisdistribution is (usually the strongest intensity) to measure the angleat which the main light path emerged from the specimen. In the last caseof FIG. 7 c, a different scanned position has caused greater refractionof the beam, which is reflected in a further shift along the array ofdetectors.

In FIG. 8, an oblong region of the specimen 6 has a higher refractiveindex (grey shape) than the rest. Rays passing around the specimen arenot refracted and so are directed to the central light detector 9 a.Rays passing through the middle of the specimen (middle two rays 11 inFIG. 8) are refracted twice. The two interfaces which the light passesthrough (white-to-grey and then grey-to-white) are parallel with eachother, and the light rays therefore exit the specimen at the same anglethat they entered it. These rays are also directed onto the centraldetector 9 a. Rays passing through other parts of the grey region arealso refracted twice but do not pass through parallel interfaces, sothese rays are detected by the adjacent light detectors 9 b.

The fact that some rays will be refracted and still exit the specimen 6parallel to the incident beam is not a problem. The example of FIG. 8shows only one of the many sets of projections taken through thissection. Full imaging involves capturing such a data set for manyorientations through the section, and the combination of all this dataallows a full reconstruction of the distribution.

FIGS. 9 to 12 show three-dimensional views of the apparatus. In FIG. 9,all un-refracted (and unscattered) rays through a two-dimensionalsection of the specimen are focused onto the central light detector ofthe array. The specimen 6 is rotated about a vertical axis betweenindexed positions in each of which a complete scan is undertaken.

FIG. 10 shows the path of scattered or refracted light onto auxiliarylight detectors.

FIG. 11 illustrates that the lens (or optical system) allows theone-dimensional array of detectors 9 to capture data from a fulltwo-dimensional raster-scan of the specimen. A row of scanned positionsis always directed down or up to the row of detectors, irrespective ofthe vertical height of the scan.

A two-dimensional array of light detectors 9 may be used instead of aone-dimensional array, as shown in FIG. 12. This would be able tomeasure light which is scattered or refracted above or below the planeoccupied by the light rays shown in FIG. 12.

In prior-art wide-field optical projection tomography, each pixel of theCCD should record the information from an approximate projection throughthe specimen. Wide-field fluorescence optical projection tomographysuffers a problem due to the fact that illumination/excitation of thespecimen must also be wide-field. If the optical properties of thespecimen cause internal scattering of light, then many photons exit thespecimen along trajectories which cause them to be detected by pixelswhich do not represent the projection from which the photon originated.This adds significant noise to the image. The light-scanning inventiondescribed here avoids this problem because only the fluorescentparticles within the approximate projection are excited at any one time.

The data derived from the detector array 9 optics is interpreted by analgorithm.

Many different algorithmic approaches already exist for performingback-projection calculations. One approach is to use a standard linearfiltered back-projection algorithm (as in U.S. Pat. No. 5,680,484).Other approaches include iterative, maximum entropy and algebraicreconstruction technique. (R. Gordon et al., “Three-DimensionalReconstruction form Projections: A Review of Algorithms”.

The algorithm works as follows:

1. The data is used as if it were parallel (or fan-beam) data to performback-projection. This produces a “fuzzy” estimation of the distributionof absorption characteristics of the specimen, or alternatively a fuzzydistribution of the fluorescence of the specimen.

2. A first approximation of the distribution of refractive index isestimated. This can be done in a number of ways. One useful method is toassume that the absorption or fluorescent distribution will reflect thedistribution of refractive index. Within each section a 2-D gradientvector is calculated for each voxel. An alternative is to start with auniform or a random distribution.

3. The estimated refraction distribution is used to perform aforward-projection, i.e. a prediction of what the projection data shouldlook like if the initial estimate of the refraction distribution wascorrect.

4. The predicted projections and the actual projections are compared.

5. The estimated refraction distribution is modified. The projectionswith a greater difference between predicted and actual, pin-point whichregions of the distribution need more modification. For example, in thecase of the grey shape shown in FIG. 8, projections from the curved endsof the oblong will differ greatly from the predictions due to the largeamount of refraction. Voxels in the regions therefore have theirpredicted refraction indexes changed more than other regions.

6. The loop from 3 to 6 is repeated until no further improvements to thepredicted projections can be made.

The algorithm approach above can also be used to interpret other opticalsignals, for example fluorescence or scattering.

It will be appreciated that modification may be made to the inventionwithout departing from the scope of the invention.

1. Apparatus for obtaining an image of a specimen by optical projectiontomography, the apparatus comprising light scanning means and a rotarystage for rotating the specimen to indexed positions in each of whichthe specimen is in use subjected to a scanning movement of incidentlight by the scanning means.
 2. Apparatus according to claim 1, whereinthe incident light is scanned in a direction perpendicular to an opticalaxis followed by the light passing through the apparatus.
 3. Apparatusaccording to claim 1, wherein the incident light is scanned in a rasterpattern, one complete scan being undertaken at each indexed position ofthe specimen.
 4. Apparatus according to claim 1, wherein the lightscanning means form part of a confocal scanning microscope.
 5. A methodof obtaining an image of a specimen by optical projection tomography,comprising scanning the specimen with a light beam and detecting lightemanating from the specimen to derive the image.
 6. A method accordingto claim 5, wherein the light passes through the specimen prior to beingdetected.
 7. A method according to claim 5, wherein the light entersfrom one side of the specimen and leaves the specimen from the same sidethereof.
 8. A method according to claim 5, wherein the specimen isrotated to indexed positions and one complete scan is undertaken at eachindexed position of the specimen.
 9. A method according to claim 5,wherein the detector detects light which exits or by-passes the specimenparallel to the beam incident on the specimen.
 10. A method according toclaim 5, wherein the light is laser light. 11-12. (canceled)